Glutarylation at Histone H4 Lysine 91 Modulates Chromatin Assembly

The best-characterized acetylation of newly synthesized histone H4 is the diacetylation of the NH2-terminal tail on lysines 5 and 12. Despite its evolutionary conservation, this pattern of modification has not been shown to be essential for either viability or chromatin assembly in any model organism. We demonstrate that mutations in histone H4 lysines 5 and 12 in yeast confer hypersensitivity to replication stress and DNA-damaging agents when combined with mutations in histone H4 lysine 91, which has also been found to be a site of acetylation on soluble histone H4. In addition, these mutations confer a dramatic decrease in cell viability when combined with mutations in histone H3 lysine 56. We also show that mutation of the sites of acetylation on newly synthesized histone H4 results in defects in the reassembly of chromatin structure that accompanies the repair of HO-mediated double-strand breaks. This defect is not due to a decrease in the level of histone H3 lysine 56 acetylation. Intriguingly, mutations that alter the sites of newly synthesized histone H4 acetylation display a marked decrease in levels of phosphorylated H2A (γ-H2AX) in chromatin surrounding the double-strand break. These results indicate that the sites of acetylation on newly synthesized histones H3 and H4 can function in nonoverlapping ways that are required for chromatin assembly, viability, and DNA damage response signaling.

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HAT4, a Golgi Apparatus-Anchored B-Type Histone Acetyltransferase, Acetylates Free Histone H4 and Facilitates Chromatin Assembly

Molecular Cell ◽

10.1016/j.molcel.2011.07.032 ◽

2011 ◽

Vol 44 (1) ◽

pp. 39-50 ◽

Cited By ~ 63

Author(s):

Xiaohan Yang ◽

Wenhua Yu ◽

Lei Shi ◽

Luyang Sun ◽

Jing Liang ◽

...

Keyword(s):

Golgi Apparatus ◽

Histone Acetyltransferase ◽

Chromatin Assembly ◽

Histone H4

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Histone acetyltransferase 1 is required for DNA replication fork function and stability

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013496 ◽

2020 ◽

Vol 295 (25) ◽

pp. 8363-8373 ◽

Cited By ~ 2

Author(s):

Paula A. Agudelo Garcia ◽

Callie M. Lovejoy ◽

Prabakaran Nagarajan ◽

Dongju Park ◽

Liudmila V. Popova ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Histone Acetyltransferase ◽

Dynamic Environment ◽

Chromatin Assembly ◽

Proximity Ligation Assay ◽

Histone H4 ◽

Replication Fork Progression ◽

Replication Fork Stalling ◽

Fork Stalling

The replisome is a protein complex on the DNA replication fork and functions in a dynamic environment at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter DNA duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the activity of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (HAT1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12, which accompanies replication-coupled chromatin assembly. Here, using proximity ligation assay-based chromatin assembly assays and DNA fiber analysis, we analyzed the role of murine HAT1 in replication-coupled chromatin assembly. We demonstrate that HAT1 physically associates with chromatin near DNA replication sites. We found that the association of HAT1 with newly replicated DNA is transient, but can be stabilized by replication fork stalling. The association of HAT1 with nascent chromatin may be functionally relevant, as HAT1 loss decreased replication fork progression and increased replication fork stalling. Moreover, in the absence of HAT1, stalled replication forks were unstable, and newly synthesized DNA became susceptible to MRE11-dependent degradation. These results suggest that HAT1 links replication fork function to the proper processing and assembly of newly synthesized histones.

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Monomethylation of Lysine 20 on Histone H4 Facilitates Chromatin Maturation

Molecular and Cellular Biology ◽

10.1128/mcb.00989-08 ◽

2008 ◽

Vol 29 (1) ◽

pp. 57-67 ◽

Cited By ~ 30

Author(s):

Annette N. D. Scharf ◽

Karin Meier ◽

Volker Seitz ◽

Elisabeth Kremmer ◽

Alexander Brehm ◽

...

Keyword(s):

Histone Modifications ◽

Chromatin Structure ◽

Chromatin Assembly ◽

Assembly System ◽

Histone H4 ◽

Dynamic Changes ◽

Histone Deposition

ABSTRACT Histone modifications play an important role in shaping chromatin structure. Here, we describe the use of an in vitro chromatin assembly system from Drosophila embryo extracts to investigate the dynamic changes of histone modifications subsequent to histone deposition. In accordance with what has been observed in vivo, we find a deacetylation of the initially diacetylated isoform of histone H4, which is dependent on chromatin assembly. Immediately after deposition of the histones onto DNA, H4 is monomethylated at K20, which is required for an efficient deacetylation of the H4 molecule. H4K20 methylation-dependent dl(3)MBT association with chromatin and the identification of a dl(3)MBT-dRPD3 complex suggest that a deacetylase is specifically recruited to the monomethylated substrate through interaction with dl(3)MBT. Our data demonstrate that histone modifications are added and removed during chromatin assembly in a highly regulated manner.

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Histone modification in early and late Drosophila embryos

Biochemical Journal ◽

10.1042/bj2180321 ◽

1984 ◽

Vol 218 (2) ◽

pp. 321-329 ◽

Cited By ~ 15

Author(s):

V Giancotti ◽

E Russo ◽

F de Cristini ◽

G Graziosi ◽

F Micali ◽

...

Keyword(s):

Histone H3 ◽

Mitotic Rate ◽

Chromatin Assembly ◽

Histone H4 ◽

Histone H2b ◽

Developmental States ◽

Histone H3 Acetylation ◽

H3 Acetylation ◽

Transcription 3 ◽

Drosophila Embryos

Levels of histone acetylation and phosphorylation have been contrasted in two developmental states of Drosophila melanogaster. The 0-2 h nuclei of the syncitial blastula are characterized by rapid mitoses and DNA replication, but there is very little transcription. In the 18 h embryo there is considerable transcription and the mitotic rate is much slower. It has been found that (1) histone H1 from 2h nuclei is not highly phosphorylated. This observation is not in accord with the view that H1 hyperphosphorylation is essential to mitosis, but is compatible with the hypothesis that H1 phosphorylation in Drosophila species is related to heterochromatization. (2) Histone H4 from 2 h embryos shows high levels of the diacetyl form (H4-Ac2), which is principally outside the nucleus. This accords with the hypothesis that H4-Ac2 is the form in which H4 is deposited on to newly replicated DNA and shows that H4 acetylation is linked not only to transcription. (3) Histone H3 acetylation is similar in 2h and in 18h embryos. As with H4, this acetylation probably correlates with chromatin assembly and is not transcription-related. (4) Histone H2B carries no modification in 2h or in 18h embryos, and H2A shows a single modification in 2h embryos and two in 18 h embryos. H2B modification is thus not essential either in mitosis or replication, whereas H2A modification is important in one or both processes. (5) The nucleosomal protein D2 is equally present in 2h and 18 h embryos.

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MUTATION ON WD DIPEPTIDE MOTIFS OF THE p48 SUBUNIT OF CHROMATIN ASSEMBLY FACTOR-1 CAUSING VIABILITY AND GROWTH OF DT40 CHICKEN B CELL LINE

Indonesian Journal of Chemistry ◽

10.22146/ijc.21468 ◽

2010 ◽

Vol 10 (2) ◽

pp. 245-250

Author(s):

Ahyar Ahmad ◽

Harningsih Karim

Keyword(s):

Cell Viability ◽

B Cell ◽

Cell Line ◽

Protein Complex ◽

Protein Family ◽

Chromatin Assembly ◽

Histone H4 ◽

Nucleosome Assembly ◽

Chromatin Assembly Factor ◽

Assembly Factor

Chromatin assembly factor-1 (CAF-1), a protein complex consisting of three subunits, p150, p60, and p48, is highly conserved from yeast to humans and facilitated nucleosome assembly of newly replicated DNA. The p48 subunit, CAF-1p48 (p48), with seven WD (Trp-Asp) repeat motifs, is a member of the WD protein family. The immunoprecipitation experiment revealed that ß-propeller structure of p48 was less stringent for it's binding to HDAC-1, but more stringent for its binding to both histones H4 and CAF-1p60 but not to ASF-1, indicating that the proper ß-propeller structure of p48 is essential for the binding to these two proteins histone H4 and CAF-1p60. Complementation experiments, involving missense and truncated mutants of FLAG-tagged p48, revealed that mutations of every of seven WD dipeptide motifs, like both the N-terminal and C-terminal truncated mutations, could not rescue for the tet-induced lethality. These results indicate not only that p48 is essential for the viability of vertebrate cells, although the yeast p48 homolog is nonessential, but also that all the seven WD dipeptide motifs are necessary for the maintenance of the proper structure of p48 that is fundamentally important for cell viability. Keywords: Chromatin assembly factor-1, complementation experiments, viability

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Histone Acetyltransferase 1 is Required for DNA Replication Fork Function and Stability

10.1101/2020.03.17.989434 ◽

2020 ◽

Author(s):

Paula A. Agudelo Garcia ◽

Callie Lovejoy ◽

Prabakaran Nagarajan ◽

Dongju Park ◽

Liudmila Popova ◽

...

Keyword(s):

Dna Replication ◽

Replication Fork ◽

Histone Acetyltransferase ◽

Dynamic Environment ◽

Chromatin Assembly ◽

Histone H4 ◽

Replication Fork Progression ◽

Replication Fork Stalling ◽

Fork Stalling ◽

Stalled Replication Forks

ABSTRACTThe replisome functions in a dynamic environment that is at the intersection of parental and nascent chromatin. Parental nucleosomes are disrupted in front of the replication fork. The daughter duplexes are packaged with an equal amount of parental and newly synthesized histones in the wake of the replication fork through the action of the replication-coupled chromatin assembly pathway. Histone acetyltransferase 1 (Hat1) is responsible for the cytosolic diacetylation of newly synthesized histone H4 on lysines 5 and 12 that accompanies replication-coupled chromatin assembly. Analysis of the role of Hat1 in replication-coupled chromatin assembly demonstrates that Hat1 also physically associates with chromatin near sites of DNA replication. The association of Hat1 with newly replicated DNA is transient but can be stabilized by replication fork stalling. The association of Hat1 with nascent chromatin may be functionally relevant as loss of Hat1 results in a decrease in replication fork progression and an increase in replication fork stalling. In addition, in the absence of Hat1, stalled replication forks are unstable and newly synthesized DNA becomes susceptible to Mre11-dependent degradation. These results suggest that Hat1 links replication fork function to the proper processing and assembly of newly synthesized histones.

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CAF-1 and Rtt101p function within the replication-coupled chromatin assembly network to promote H4 K16ac, preventing ectopic silencing

PLoS Genetics ◽

10.1371/journal.pgen.1009226 ◽

2020 ◽

Vol 16 (12) ◽

pp. e1009226

Author(s):

Tiffany J. Young ◽

Yi Cui ◽

Claire Pfeffer ◽

Emilie Hobbs ◽

Wenjie Liu ◽

...

Keyword(s):

Replication Fork ◽

Chromatin Modification ◽

Chromatin Assembly ◽

Histone H4 ◽

Alternate Pathway ◽

Major Implication ◽

Assembly Factor ◽

Histone Modifying Enzymes ◽

Assembly Pathways ◽

Dependent Pathway

Replication-coupled chromatin assembly is achieved by a network of alternate pathways containing different chromatin assembly factors and histone-modifying enzymes that coordinate deposition of nucleosomes at the replication fork. Here we describe the organization of a CAF-1-dependent pathway in Saccharomyces cerevisiae that regulates acetylation of histone H4 K16. We demonstrate factors that function in this CAF-1-dependent pathway are important for preventing establishment of silenced states at inappropriate genomic sites using a crippled HMR locus as a model, while factors specific to other assembly pathways do not. This CAF-1-dependent pathway required the cullin Rtt101p, but was functionally distinct from an alternate pathway involving Rtt101p-dependent ubiquitination of histone H3 and the chromatin assembly factor Rtt106p. A major implication from this work is that cells have the inherent ability to create different chromatin modification patterns during DNA replication via differential processing and deposition of histones by distinct chromatin assembly pathways within the network.

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Mechanisms Underlying Acrolein-Mediated Inhibition of Chromatin Assembly

Molecular and Cellular Biology ◽

10.1128/mcb.00448-16 ◽

2016 ◽

Vol 36 (23) ◽

pp. 2995-3008 ◽

Cited By ~ 9

Author(s):

Lei Fang ◽

Danqi Chen ◽

Clinton Yu ◽

Hongjie Li ◽

Jason Brocato ◽

...

Keyword(s):

Fluorescent Protein ◽

Chromatin Assembly ◽

Histone H4 ◽

Nucleosome Assembly ◽

Histone Lysine ◽

Lysine Residues ◽

Underlying Mechanisms ◽

Green Fluorescent

Acrolein is a major component of cigarette smoke and cooking fumes. Previously, we reported that acrolein compromises chromatin assembly; however, underlying mechanisms have not been defined. Here, we report that acrolein reacts with lysine residues, including lysines 5 and 12, sites important for chromatin assembly, on histone H4 in vitro and in vivo . Acrolein-modified histones are resistant to acetylation, suggesting that the reduced H4K12 acetylation that occurs following acrolein exposure is probably due to the formation of acrolein-histone lysine adducts. Accordingly, the association of H3/H4 with the histone chaperone ASF1 and importin 4 is disrupted and the translocation of green fluorescent protein-tagged H3 is inhibited in cells exposed to acrolein. Interestingly, in vitro plasmid supercoiling assays revealed that treatment of either histones or ASF1 with acrolein has no effect on the formation of plasmid supercoiling, indicating that acrolein-protein adduct formation itself does not directly interfere with nucleosome assembly. Notably, exposure of histones to acrolein prior to histone acetylation leads to the inhibition of remodeling and spacing factor chromatin assembly, which requires acetylated histones for efficient assembly. These results suggest that acrolein compromises chromatin assembly by reacting with histone lysine residues at the sites critical for chromatin assembly and prevents these sites from physiological modifications.

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Yeast Chromatin Assembly Complex 1 Protein Excludes Nonacetylatable Forms of Histone H4 from Chromatin and the Nucleus

Molecular and Cellular Biology ◽

10.1128/mcb.24.23.10180-10192.2004 ◽

2004 ◽

Vol 24 (23) ◽

pp. 10180-10192 ◽

Cited By ~ 35

Author(s):

Lynn Glowczewski ◽

Jakob H. Waterborg ◽

Judith G. Berman

Keyword(s):

Nuclear Localization ◽

Nuclear Import ◽

Fluorescent Protein ◽

Chromatin Assembly ◽

Histone H4 ◽

Wild Type ◽

Acetylation State ◽

Assembly Factor ◽

Heterochromatin Silencing ◽

Green Fluorescent

ABSTRACT In yeast, the establishment and maintenance of a transcriptionally silent chromatin state are dependent upon the acetylation state of the N terminus of histone proteins. Histone H4 proteins that contain mutations in N-terminal lysines disrupt heterochromatin and result in yeast that cannot mate. Introduction of a wild-type copy of histone H4 restores mating, despite the presence of the mutant protein, suggesting that mutant H4 protein is either excluded from, or tolerated in, chromatin. To understand how the cell differentiates wild-type histone and mutant histone in which the four N-terminal lysines were replaced with alanine (H4-4A), we analyzed silencing, growth phenotypes, and the histone composition of chromatin in yeast strains coexpressing equal amounts of wild-type and mutant H4 proteins (histone H4 heterozygote). We found that histone H4 heterozygotes have defects in heterochromatin silencing and growth, implying that mutations in H4 are not completely recessive. Nuclear preparations from histone H4 heterozygotes contained less mutant H4 than wild-type H4, consistent with the idea that cells exclude some of the mutant histone. Surprisingly, the N-terminal nuclear localization signal of H4-4A fused to green fluorescent protein was defective in nuclear localization, while a mutant in which the four lysines were replaced with arginine (H4-4R) appeared to have normal nuclear import, implying a role for the charged state of the acetylatable lysines in the nuclear import of histones. The biased partial exclusion of H4-4A was dependent upon Cac1p, the largest subunit of yeast chromatin assembly factor 1 (CAF-1), as well as upon the karyopherin Kap123p, but was independent of Cac2p, another CAF-1 component, and other chromatin assembly proteins (Hir3p, Nap1p, and Asf1p). We conclude that N-terminal lysines of histone H4 are important for efficient histone nuclear import. In addition, our data support a model whereby Cac1p and Kap123 cooperate to ensure that only appropriately acetylated histone H4 proteins are imported into the nucleus.

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